This invention relates to centrifugal methods and devices for concentrating macromolecules from a solution without filtering to dryness and for recovering a maximal amount of concentrated macromolecular retentate.
There have been a number of analytical procedures developed in the biochemical art wherein it is required to remove solvent from protein solutions in order to have a more concentrated protein sample which can be analyzed effectively, or in order to have a protein-free filtrate for analysis, or to replace or remove low molecular weight ions or solutes, or to study protein-binding attributes of various chemicals in combination with various protein samples. Many other analytical procedures, involving not only proteins but macromolecular species in general, have also been developed wherein it is necessary to concentrate a macromolecular component in a liquid sample.
When concentrating small volumes of macromolecules in solution using filtration, e.g., ultrafiltration, there exists the problem of filtration to dryness. Although a fixed volume of buffer may be added to a microvolume concentration device to redissolve macromolecules retained after filtration to dryness, it is generally found that total mass recovery of macromolecules is significantly less, and biological activity recovery is often reduced, as compared to the case when filtration is stopped at the desired final retentate volume.
In the prior art, the Amicon Minicon.RTM. adsorbent activated concentrator is provided with an impermeable seal coating on the portion of the membrane in contact with the desired final retentate volume. This coating impedes filtration to dryness, but does not prevent it, due to wicking caused by surface tension forces, which allows continued filtration after the retentate meniscus recedes onto the coated area.
The centrifugal microconcentrator of this invention is of similar design to the Amicon MPS-1 Micropartition System (Publication 472). In the MPS-1, however, the membrane support is provided with filtrate ducts under all portions of the membrane, so that if left unattended while being centrifuged in a fixed angle rotor, the MPS-1 will filter to dryness.
Besides the problem of filtration to dryness, there also exists the problem of recovery of microvolumes of concentrated macromolecular retentate from a microconcentrator. Recovery with a conventional pipette is difficult due to film losses caused by surface tension wetting of the membrane and reservoir walls. Also, the pipette tip may damage the membrane skin, preventing optional reuse of the microconcentrator. For example, recovery of retentate with a pipette from the centrifugal self-cleaning ultrafilter, described in U.S. Pat. No. 3,488,768, the teachings of which are incorporated herein, is limited due to the large membrane surface area which causes adsorptive and film loss of the macromolecules.
Accordingly, it is a principal object of this invention to provide a centrifugal microconcentrator which can concentrate macromolecules from a solution without filtering to dryness.
It is another object of this invention to provide such a microconcentrator which gives constant final retentate volume, for a given fixed rotor angle, regardless of the alignment of the device in the rotor.
It is another object of this invention to use such a concentrator in a centrifugal recovery process in which a maximal amount of concentrated macromolecular retentate is obtained without damage to the membrane and with minimal film loss.
It is another object of this invention to provide a centrifugal recovery process for the recovery of a maximal amount of concentrated macromolecular retentate from a centrifugal concentrator device.
It is a further object of this invention to provide a reuseable centrifugal microconcentrator.
It is a still further object of this invention to provide a centrifugal microvolume concentrator that is economic to manufacture.